Neuronal & Endothelial NO synthase enzymes (nNOS & eNOS) make NO in response to calmodulin (CaM) binding and function broadly in human health and disease. Our long-term goal is to understand the molecular mechanisms that regulate NOS catalysis. NOS contain connected NADPH-FAD (FNR), FMN, and NOSoxy domains that transfer NADPH-derived electrons to the heme and tetrahydrobiopterin (H4B) groups in NOSoxy, thus allowing O2 activation required for NO synthesis. We believe that NOS conformational states and domain motions control its electron transfer reactions. The FMN domain plays a key role by performing separate electron acceptor and donor functions to shuttle electrons through NOS. This creates a three-state, two- equilibrium model that involves separate FMN domain interactions with the FNR (KA) and with the NOSoxy (KB). We hypothesize that structural & conformational features that are common to di-flavin oxidoreductases blend with those unique to NOS enzymes to create novel mechanisms of regulation. How the elements function at the molecular level to regulate NOS conformational behaviors and synchronize electron transfer reactions is a central focus of this proposal. During the current funding period we developed methods to: a) Study KA & KB in nNOS & eNOS b) Simulate their different rates of conformational change & electron flux, and c) Characterize several common & unique structural elements that control their electron transfers. Despite significant progress, connections between NOS conformational states & motions and the electron transfer reactions remain largely unexplored. Our Aims address this gap through connected biophysical, biochemical, kinetic, and molecular engineering approaches, to achieve a molecular-level understanding of NOS regulation. Aim 1. Define conformational KA, its control mechanisms, and its role in determining electron flux through nNOSr & eNOSr. We will: Utilize our newly-developed Cys-lite, FRET, & domain locking approaches to: (i) define conformational distributions & populations associated with KA, & test hypotheses regarding control by the flavin reduction state & CaM binding. (ii) Define how the common & unique control elements in NOS orchestrate KA and FMN domain motions to uncover molecular mechanisms regulating electron flux. Aim 2. What mechanisms govern KB & the associated FMN to NOSoxy electron transfer? We will use our Cys-lite, FRET, & domain locking approaches to: (i) define conformational states and distributions associated with KB that underpin FMN to heme electron transfer, (ii) Determine how conformational properties are influenced by the flavin & heme redox state, CaM binding, and control elements that regulate heme reduction, and (iii) Test whether these same control mechanisms regulate electron transfer to H4B in nNOS. Relevance: By clarifying how NO production is regulated at the enzyme level our work may help develop treatments for human diseases that involve making too much or too little NO, and will illuminate protein structure-function relationships among this important class of redox proteins.